1. Field of the Invention
This invention relates to immunoassay of an analyte and materials used therein, and more particularly relates to a method and materials for enzyme immunoassay which includes multiple binding reactions.
2. Background of the Invention
A variety of assay systems which are both rapid and sensitive has been developed to detect or determine the concentration of a substance, generally referred to as the analyte, in a liquid. Immunoassays depend on the binding of the analyte to a specific antianalyte, and have been particularly useful because they give high levels of specificity and sensitivity. These assays generally employ one of the above reagents in labeled form, the labeled reagent often being referred to as the tracer. Immunoassay procedures may be carried out in solution or on a solid support, and are of two basic types. In competitive assays, the tracer is labeled analyte, and the analyte and tracer compete for a limited number of antianalyte binding sites. In sandwich assays, the tracer is a labeled second antianalyte specific for a second determinant on the analyte giving an antianalyte-analyte-labeled antianalyte sandwich.
Various means for labeling have been developed. Radioimmunoassay (RIA) procedures use radioisotopes as labels, provide high levels of sensitivity and reproducibility, and are amenable to automation for rapid processing of large numbers of samples. However, all RIA procedures require a separation step, since the parameter measured (nuclear decay) cannot be controlled by changing assay conditions or components. In addition, isotopes are costly, have relatively short shelf lives, require expensive and complex equipment, and extensive safety measures for their handling and disposal must be followed.
Fluoroimmunoassay (FIA) uses fluorochromes as labels, provides direct detection of the label, and is readily adaptable to homogeneous assay procedures. However, known homogeneous FIA methods using organic fluorochromes, such as fluorescein or rhodamine derivatives, have not achieved the high sensitivity of RIA, largely because of light scattering by impurities suspended in the assay medium and by background fluorescence emission from other fluorescent materials present in the assay medium.
Enzymes have also been used as labels in immunoassay. Enzyme immunoassay (EIA) combines the advantages of RIA and FIA and overcomes many of the disadvantages of the other two methods. Enzyme labeled reagents are cheap to prepare and are highly stable thus giving a long shelf life, yet yield assays which approach the sensitivity of radioimmunoassay and which give objective results that can be determined either visually or with rather simple equipment, such as a spectrophotometer.
In conventional EIA, an enzyme is covalently conjugated with one component of a specifically binding antigen-antibody pair, and the resulting enzyme conjugate is reacted with a substrate to produce a color which is measured. Often, an unconjugated component is immobilized on a solid support. Representative of such conventional EIA is U.S. Pat. No. 3,654,090 to Schuurs et al.
Analytes to be determined in biological fluids are often present in the range of 10.sup.-9 to 10.sup.-12 M, and conventional EIA techniques may not be sufficiently sensitive to detect analytes present in such low concentrations. In the art, EIA sensitivity has been increased by cascade amplification in which the number of detectable (generally colored) molecules is increased by use of multiple enzymes or enzyme derivatives. In this procedure, a first enzyme conjugated to an assay component may activate a second enzyme or enzyme derivative which catalyzes a color producing reaction or formation of a third enzyme. Exemplary of this technique is U.S. Pat. No. 4,463,090 to Harris.
U.S. Pat. No. 4,446,231 to Self discloses a cyclying amplification enzyme immunoassay which includes primary and secondary enzyme systems and a modulator for the second enzyme system. The primary system includes a first enzyme coupled to a ligand. In a first embodiment of the Self invention, the first enzyme system acts on a modulator precursor to liberate a modulator. The modulator is a cofactor of the secondary enzyme which activates the second enzyme system to catalyze the reaction of a substrate to a detectable product. During the reaction, the modulator is converted to an inactive form, and cycling is accomplished by a third enzyme which reactivates the modulator. In a second embodiment, the modulator is an inhibitor of the secondary system and is removed by the primary enzyme system whereby the secondary system is activated to act on the substrate and thereby produce the detectable product.
A homogenous competitive EIA in which an analyte-cytolysin conjugate binds competitively to a specific antianalyte is disclosed by Freytag et al. in U.S. Pat. No. 4,517,303. The cytolysin component of unbound conjugate ruptures vesicles containing a sequestered marker.
U.S. Pat. No. 4,543,325 to Albert et al. discloses a method for immunoassay of creatinine. 1-Methylhydantoin formed enzymatically in the assay fluid competes with enzyme-labeled 1-methylhydantoin for a limited quantity of anti-1-methylhydantoin antibody on a solid phase.